Crystallographic and thermodynamic evidence of negative cooperativity of flavin and tryptophan binding in the flavin-dependent halogenases AbeH and BorH.

The flavin-dependent halogenase AbeH produces 5-chlorotryptophan in the biosynthetic pathway of the chlorinated bisindole alkaloid BE-54017. We report that in vitro, AbeH (assisted by the flavin reductase AbeF) can chlorinate and brominate tryptophan as well as other indole derivatives and substrates with phenyl and quinoline groups. We solved the X-ray crystal structures of AbeH alone and complexed with FAD, as well as crystal structures of the tryptophan-6-halogenase BorH alone, in complex with 6-chlorotryptophan, and in complex with FAD and tryptophan. Partitioning of FAD and tryptophan into different chains of BorH and failure to incorporate tryptophan into AbeH/FAD crystals suggested that flavin and tryptophan binding are negatively coupled in both proteins. ITC and fluorescence quenching experiments confirmed the ability of both AbeH and BorH to form binary complexes with FAD or tryptophan and the inability of tryptophan to bind to AbeH/FAD or BorH/FAD complexes. FAD could not bind to BorH/tryptophan complexes, but FAD appears to displace tryptophan from AbeH/tryptophan complexes in an endothermic entropically-driven process.

Steady-state kinetic analysis of halogenase-supporting flavin reductases BorF and AbeF reveals different kinetic mechanisms.

The short-chain flavin reductases BorF and AbeF reduce FAD to FADH2, which is then used by flavin-dependent halogenases (BorH and AbeH respectively) to regioselectively chlorinate tryptophan in the biosynthesis of indolotryptoline natural products. Recombinant AbeF and BorF were overexpressed and purified as homodimers from E. coli, and copurified with substoichiometric amounts of FAD, which could be easily removed. AbeF and BorF can reduce FAD, FMN, and riboflavin in vitro and are selective for NADH over NADPH. Initial velocity studies in the presence and absence of inhibitors showed that BorF proceeds by a sequential ordered kinetic mechanism in which FAD binds first, while AbeF follows a random-ordered sequence of substrate binding. Fluorescence quenching experiments verified that NADH does not bind BorF in the absence of FAD, and that both AbeF and BorF bind FAD with higher affinity than FADH2. pH-rate profiles of BorF and AbeF were bell-shaped with maximum kcat at pH 7.5, and site-directed mutagenesis of BorF implicated His160 and Arg38 as contributing to the catalytic activity and the pH dependence.